CD200 (OX2) is a membrane glycoprotein that interacts with a structurally related receptor (CD200... more CD200 (OX2) is a membrane glycoprotein that interacts with a structurally related receptor (CD200R) involved in the regulation of macrophage function. The interaction is of low affinity (KD ∼ 1 μm) but can be detected using CD200 displayed in a multivalent form on beads or with dimeric fusion proteins consisting of the extracellular region of CD200 and immunoglobulin Fc regions. We prepared putative pentamers and trimers of mouse CD200 with sequences from cartilage oligomeric matrix protein (COMP) and surfactant protein D (SP-D), respectively. The COMP protein gave high-avidity binding and was a valuable tool for showing the interaction whilst the SP-D protein gave weak binding. In vivo experiments showed that an agonistic CD200R monoclonal antibody caused some amelioration in a model of experimental autoimmune encephalomyelitis but the COMP protein was cleared rapidly and had minimal effect. Pentameric constructs also allowed detection of the rat CD48/CD2 interaction, which is of much lower affinity (KD ∼ 70 μm). These reagents may have an advantage over Fc-bearing hybrid molecules for probing cell surface proteins without side-effects due to the Fc regions. The CD200-COMP gave strong signals in protein microarrays, suggesting that such reagents may be valuable in high throughput detection of weak interactions.
Journal of Neuropathology and Experimental Neurology, Nov 1, 1991
The CD44 antigen is a proteoglycan recently implicated in several adhesion events including that ... more The CD44 antigen is a proteoglycan recently implicated in several adhesion events including that of lymphocytes to endothelium. The CD44 antigen, reactive with monoclonal antibody (MAb) 44D10, has been shown previously to be expressed in normal human white matter homogenates and to be found at higher concentrations in brain homogenates of victims of multiple sclerosis (MS). The cellular localization of CD44 in human brain of normal individuals and in those afflicted with MS has now been determined. Monoclonal antibody 44D10 reacted with astrocyte-like cells in 40 microns thick paraformaldehyde-fixed sections but not in thin (6 microns) fixed sections. A double labeling experiment performed on a frozen brain section with MAb 44D10 and rabbit anti-glial fibrillary acidic protein (GFAP), a cytoplasmic marker of astrocytes, confirmed the co-localization of these two antigens. The reactivity with brain tissue sections of a rabbit antiserum produced against lymphocyte-CD44 could be absorbed by a preparation of the CD44 glycoprotein, purified 2,100-fold from a white matter homogenate. The antiserum was shown by Western blot analysis to be specific for p80 glycoprotein in brain extracts derived from a normal and MS patients. This antibody reacted with fibrous astrocytes predominantly in white matter; staining was also noted in subependymal and subpial regions. Inhibition studies using a cellular radioimmunoassay indicated that the highest concentrations of CD44 in three MS victims were found in plaques, followed by periplaques and non-involved areas of white matter which were higher than normal white matter. Reactive astrocytes, identified in active lesions, expressed high levels of CD44 on their surfaces. Thus, CD44 is associated with astrocytes in human brain and the increased expression observed in MS brain may reflect activation and/or proliferation of astrocytes implicated in the pathogenesis of this disease.
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a variety of phenotypic cha... more The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a variety of phenotypic changes on normal and malignant cells. In chronic and acute lymphocytic leukemia we have observed a marked augmentation in mixed lymphocyte reaction stimulatory capacity (MLRs) following pretreatment of leukemia blasts with TPA. We have now characterized the effects of TPA on MLRs utilizing the non-T, non-B lymphoblastic leukemia-cell line, HOON. Following pretreatment with TPA (optimal concentration, 1.6 X 10(-10) M), but not with other phorbol esters, there was a marked increase in MLRs, particularly at lower stimulator-cell concentrations. This effect was maximal following a 44-hr preincubation period and the enhancement in MLRs was not accompanied by changes in levels of Ia expression. This cell line provides a model for determining the molecular basis for the TPA-induced augmentation of stimulatory capacity in the mixed lymphocyte reaction.
The number of Ia molecules accessible at the surface of a cell could be related to the stage of d... more The number of Ia molecules accessible at the surface of a cell could be related to the stage of differentiation and to the functional status of that cell. We have measured the level of Ia expressed on various normal and leukemic human lymphocytes using monoclonal antibody 21w4 which recognizes a non-polymorphic epitope of human Ia (1,2). Our results with ALL and CLL do not support a correlation between stage of differentiation and level of Ia. Furthermore PHA-activated T cells and tonsillar B cells express the highest amounts of Ia.
Publisher Summary This chapter explores the θ antigen, which is designated as Thy-1 antigen. The ... more Publisher Summary This chapter explores the θ antigen, which is designated as Thy-1 antigen. The two alleles of the Thy-1 gene are referred to as Thy-1a and Thy-1b. The product encoded by the Thy-1a allele is the Thy-l.1 alloantigen (θ-AKR) and the product of the Thy-1b allele is the Thy-l.2 alloantigen (θ-C3H). It discusses the distribution of Thy-l.l and Thy-l.2 alloantigens in different murine strains. The chapter reviews that congenic lines differing at the Thy-1 locus have been developed: A- Thy-1a and A.AL-old carrying the Thy-1a allele on A background and B10.PL carrying the Thy-1a allele on the B10 background. Such congenic lines have established the Thy-1.1 and Thy-l.2 allospecificities of allosera and of monoclonal antibodies. It discusses that Thy-1.1 and Thy-l.2 antigens are present in large amounts in brain and thymus of mice but in smaller amounts on thymus-derived peripheral T cells. Lymph node, spleen, and bone marrow cell suspension have anti-Thy-1 absorptive capacities of 14, 7, and 1% relative to thymocytes. It also reviews that Thy-1 is a general T cell marker only in the murine species. The appearance of Thy-1 on mouse lymphocytes is generally associated with maturation in the thymus. Thymectomized and nude mice show a marked reduction in the number of Thy-1 bearing cells in lymph nodes and spleen. In thymic nude mice, however, immunocompetent cells expressing low levels of Thy-1 are present.
A.TH anti-A.TL alloserum has previously been shown to react with monomorphic determinants of huma... more A.TH anti-A.TL alloserum has previously been shown to react with monomorphic determinants of human Ia molecules. In an attempt to produce monoclonal antibodies detecting human-mouse cross-reacting epitopes, A.TH mice (Is) which lack I-E antigens were immunized with human Ia glycoproteins. Five hybridoma lines were established which recognize monomorphic human Ia determinants. Only one of the lines, 21w4, is reactive with murine cells and also with pig and sheep cells but not with rat cells. Binding studies to murine target cells show a strain distribution analogous to that of an Ia. 7-like specificity. The binding of 21w4 antibody to human and murine cells was compared to that of two other monoclonal antibodies directed to the murine Ia. 7-like specificity. Although all three antibodies had a similar pattern of reactivity with cells of different mouse strains, only 21w4 bound to the human cells tested. Competitive inhibition studies on murine cells have revealed that the epitopes were spatially related but not identical. Our results support the view that Ia. 7, the putative species cross-reacting specificity, might be composed of clusters of epitopes with variable degrees of conservation throughout evolution.
The concentration of a glycoprotein reactive with monoclonal antibody 44D10 was studied during de... more The concentration of a glycoprotein reactive with monoclonal antibody 44D10 was studied during development and aging in 19 normal brains. Little change in the concentration of the antigen was found in white matter of brains ranging from the age of 1 to 54 years. However, a linear increase in the concentration of antigen was observed between the ages of 54 and 80 years. By the age of 80 years, the concentration of the glycoprotein had increased 2-3-fold. In contrast to white matter, gray matter did not contain detectable levels of antigen at ages from 1 to 54 years. Low levels of antigen were detected in brains of all older individuals examined. However, the relative concentration of this glycoprotein in gray matter was less than 5% of that in white matter. An examination of white matter homogenates from guinea pig and bovine brain showed that monoclonal antibody 44D10 was not reactive in these species.
CD200 (OX2) is a membrane glycoprotein that interacts with a structurally related receptor (CD200... more CD200 (OX2) is a membrane glycoprotein that interacts with a structurally related receptor (CD200R) involved in the regulation of macrophage function. The interaction is of low affinity (KD ∼ 1 μm) but can be detected using CD200 displayed in a multivalent form on beads or with dimeric fusion proteins consisting of the extracellular region of CD200 and immunoglobulin Fc regions. We prepared putative pentamers and trimers of mouse CD200 with sequences from cartilage oligomeric matrix protein (COMP) and surfactant protein D (SP-D), respectively. The COMP protein gave high-avidity binding and was a valuable tool for showing the interaction whilst the SP-D protein gave weak binding. In vivo experiments showed that an agonistic CD200R monoclonal antibody caused some amelioration in a model of experimental autoimmune encephalomyelitis but the COMP protein was cleared rapidly and had minimal effect. Pentameric constructs also allowed detection of the rat CD48/CD2 interaction, which is of much lower affinity (KD ∼ 70 μm). These reagents may have an advantage over Fc-bearing hybrid molecules for probing cell surface proteins without side-effects due to the Fc regions. The CD200-COMP gave strong signals in protein microarrays, suggesting that such reagents may be valuable in high throughput detection of weak interactions.
Journal of Neuropathology and Experimental Neurology, Nov 1, 1991
The CD44 antigen is a proteoglycan recently implicated in several adhesion events including that ... more The CD44 antigen is a proteoglycan recently implicated in several adhesion events including that of lymphocytes to endothelium. The CD44 antigen, reactive with monoclonal antibody (MAb) 44D10, has been shown previously to be expressed in normal human white matter homogenates and to be found at higher concentrations in brain homogenates of victims of multiple sclerosis (MS). The cellular localization of CD44 in human brain of normal individuals and in those afflicted with MS has now been determined. Monoclonal antibody 44D10 reacted with astrocyte-like cells in 40 microns thick paraformaldehyde-fixed sections but not in thin (6 microns) fixed sections. A double labeling experiment performed on a frozen brain section with MAb 44D10 and rabbit anti-glial fibrillary acidic protein (GFAP), a cytoplasmic marker of astrocytes, confirmed the co-localization of these two antigens. The reactivity with brain tissue sections of a rabbit antiserum produced against lymphocyte-CD44 could be absorbed by a preparation of the CD44 glycoprotein, purified 2,100-fold from a white matter homogenate. The antiserum was shown by Western blot analysis to be specific for p80 glycoprotein in brain extracts derived from a normal and MS patients. This antibody reacted with fibrous astrocytes predominantly in white matter; staining was also noted in subependymal and subpial regions. Inhibition studies using a cellular radioimmunoassay indicated that the highest concentrations of CD44 in three MS victims were found in plaques, followed by periplaques and non-involved areas of white matter which were higher than normal white matter. Reactive astrocytes, identified in active lesions, expressed high levels of CD44 on their surfaces. Thus, CD44 is associated with astrocytes in human brain and the increased expression observed in MS brain may reflect activation and/or proliferation of astrocytes implicated in the pathogenesis of this disease.
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a variety of phenotypic cha... more The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a variety of phenotypic changes on normal and malignant cells. In chronic and acute lymphocytic leukemia we have observed a marked augmentation in mixed lymphocyte reaction stimulatory capacity (MLRs) following pretreatment of leukemia blasts with TPA. We have now characterized the effects of TPA on MLRs utilizing the non-T, non-B lymphoblastic leukemia-cell line, HOON. Following pretreatment with TPA (optimal concentration, 1.6 X 10(-10) M), but not with other phorbol esters, there was a marked increase in MLRs, particularly at lower stimulator-cell concentrations. This effect was maximal following a 44-hr preincubation period and the enhancement in MLRs was not accompanied by changes in levels of Ia expression. This cell line provides a model for determining the molecular basis for the TPA-induced augmentation of stimulatory capacity in the mixed lymphocyte reaction.
The number of Ia molecules accessible at the surface of a cell could be related to the stage of d... more The number of Ia molecules accessible at the surface of a cell could be related to the stage of differentiation and to the functional status of that cell. We have measured the level of Ia expressed on various normal and leukemic human lymphocytes using monoclonal antibody 21w4 which recognizes a non-polymorphic epitope of human Ia (1,2). Our results with ALL and CLL do not support a correlation between stage of differentiation and level of Ia. Furthermore PHA-activated T cells and tonsillar B cells express the highest amounts of Ia.
Publisher Summary This chapter explores the θ antigen, which is designated as Thy-1 antigen. The ... more Publisher Summary This chapter explores the θ antigen, which is designated as Thy-1 antigen. The two alleles of the Thy-1 gene are referred to as Thy-1a and Thy-1b. The product encoded by the Thy-1a allele is the Thy-l.1 alloantigen (θ-AKR) and the product of the Thy-1b allele is the Thy-l.2 alloantigen (θ-C3H). It discusses the distribution of Thy-l.l and Thy-l.2 alloantigens in different murine strains. The chapter reviews that congenic lines differing at the Thy-1 locus have been developed: A- Thy-1a and A.AL-old carrying the Thy-1a allele on A background and B10.PL carrying the Thy-1a allele on the B10 background. Such congenic lines have established the Thy-1.1 and Thy-l.2 allospecificities of allosera and of monoclonal antibodies. It discusses that Thy-1.1 and Thy-l.2 antigens are present in large amounts in brain and thymus of mice but in smaller amounts on thymus-derived peripheral T cells. Lymph node, spleen, and bone marrow cell suspension have anti-Thy-1 absorptive capacities of 14, 7, and 1% relative to thymocytes. It also reviews that Thy-1 is a general T cell marker only in the murine species. The appearance of Thy-1 on mouse lymphocytes is generally associated with maturation in the thymus. Thymectomized and nude mice show a marked reduction in the number of Thy-1 bearing cells in lymph nodes and spleen. In thymic nude mice, however, immunocompetent cells expressing low levels of Thy-1 are present.
A.TH anti-A.TL alloserum has previously been shown to react with monomorphic determinants of huma... more A.TH anti-A.TL alloserum has previously been shown to react with monomorphic determinants of human Ia molecules. In an attempt to produce monoclonal antibodies detecting human-mouse cross-reacting epitopes, A.TH mice (Is) which lack I-E antigens were immunized with human Ia glycoproteins. Five hybridoma lines were established which recognize monomorphic human Ia determinants. Only one of the lines, 21w4, is reactive with murine cells and also with pig and sheep cells but not with rat cells. Binding studies to murine target cells show a strain distribution analogous to that of an Ia. 7-like specificity. The binding of 21w4 antibody to human and murine cells was compared to that of two other monoclonal antibodies directed to the murine Ia. 7-like specificity. Although all three antibodies had a similar pattern of reactivity with cells of different mouse strains, only 21w4 bound to the human cells tested. Competitive inhibition studies on murine cells have revealed that the epitopes were spatially related but not identical. Our results support the view that Ia. 7, the putative species cross-reacting specificity, might be composed of clusters of epitopes with variable degrees of conservation throughout evolution.
The concentration of a glycoprotein reactive with monoclonal antibody 44D10 was studied during de... more The concentration of a glycoprotein reactive with monoclonal antibody 44D10 was studied during development and aging in 19 normal brains. Little change in the concentration of the antigen was found in white matter of brains ranging from the age of 1 to 54 years. However, a linear increase in the concentration of antigen was observed between the ages of 54 and 80 years. By the age of 80 years, the concentration of the glycoprotein had increased 2-3-fold. In contrast to white matter, gray matter did not contain detectable levels of antigen at ages from 1 to 54 years. Low levels of antigen were detected in brains of all older individuals examined. However, the relative concentration of this glycoprotein in gray matter was less than 5% of that in white matter. An examination of white matter homogenates from guinea pig and bovine brain showed that monoclonal antibody 44D10 was not reactive in these species.
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